improved lipid accumulation through metabolic …...transformants generated this way will not be...
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Hildebrand Lab
Scripps Institution of Oceanography, University of California, San Diego
Improved Lipid Accumulation Through Metabolic
Engineering of Diatoms
Aubrey Davis
Diatoms and Lipid Accumulation
• Amongst unicellular algae, diatoms are one of the most productive classes, they
naturally grow under bloom conditions (maximum rate and biomass
accumulation in a short time period) and generally out-compete other
algae for growth (Brzezinski et al. 2001. Deep Sea Res. 48:3997; Carter et al., 2005, J. Exp. Mar. Biol. Ecol. 326:1).
• Diatoms are capable of abundant accumulation of neutral lipids (30-45%).
• Diatoms were a model organism in the Aquatic Species Program, out of a list of
50 strains selected from a pool of over 3,000 candidates that held the most promise
as biofuel production strains, 60% were diatoms (www.nrel.gov/docs/legosti/fy98/24190.pdf).
Thalassiosira Cyclotella pseudonana cryptica
Falkowski and Oliver 2007
Fundamental Analysis of Short-term Lipid Accumulation Resulting from Silicon Limitation in T. pseudonana
Sarah Smith
Fatty Acids
Photosynthesis
Whole-Genome mRNA Expression Using Microarrays
Lipid Induced
• RNA isolated from 6 timepoints (t=0h, t=4h, t=8h, t=12h, t=18h, t=24h)
• Affymetrix whole-genome tiling
microarray in duplicate (Andy Allen, JCVI)
• Bioinformatics (11,782 genes)
(Trina Norden-Krichmar, JCVI & Steve Federowicz, UCSD)
The Key Issue in Producing Useful Biomolecules: Carbon Flux
Regulating Carbon Partitioning is Required to Increase Production of Desired Biomolecules
Elucidation of Carbon Flux Processes:
Transient Induction of Gluconeogensis Genes
Sarah Smith
Storage Carbohydrate Genes
Key Finding:
The initial response to Si starvation is induction of
gluconeogenesis and carbohydrate storage,
followed later by TAG accumulation.
General Carbon Flux Response During Si Limitation
TAG
Knockdown of a Chrysolaminarin Synthase Gene
Improves TAG Accumulation
Three genes encoding steps in
chrysolaminarin synthesis are transiently
upregulated during silicon starvation
Several 12695 knockdown strains exhibit
improved TAG accumulation
The best knockdowns resulted in 1.5-3 fold
improvement in TAG levels over wild-type.
We are characterizing knockdowns in the two other
genes and generating simultaneous knockdowns in
multiple genes.
Key Finding:
Inhibition of carbon flux to a competing pathway
(carbohydrate storage) improves TAG
accumulation, in some cases without detriment to
growth.
Most strains grow as well as wild-type
Kalpana Manandhar-Shrestha
Overexpression of a DGAT Improves TAG Accumulation
Overexpression of DGAT resulted in 1.2-1.5 fold improvement in TAG levels over wild-type.
We are characterizing other enzymes involved in TAG synthesis and have generated overexpression strains.
Kalpana Manandhar-Shrestha
Characterization of CGI-58 Lipase Homologs Emily Trentacoste, Jennifer Hull, Bill Gerwick
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Thaps3_264297
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TAG
Catabolism
CGI-58 knockdowns in Arabidopsis and mutations in humans resulted in high TAG accumulation
Characterization of CGI-58 Lipase Homolog Knockdowns Emily Trentacoste, Jennifer Hull, Bill Gerwick
Total Lipid TAG Polar Lipid
Growth Curves BODIPY
Key Finding: CGI-58 homolog knockdowns improve both TAG and polar lipid
content with (in antisense cases) no detrimental affect on growth.
TAG was increased 1.9 - 4 fold
TAG TAG TAG
Catabolism
A Variety of Manipulations Lead to Increased TAG
Flexibility in choosing gene targets could minimize detrimental effects
and maintain fast growth and high biomass accumulation
Redirecting
carbon flux
Increasing
TAG synthesis Inhibiting lipid
catabolism
1.5-3 fold
improvement
1.2-1.5 fold
improvement
1.9-4 fold
improvement
Mitochondrion Cytoplasm Chloroplast
There are More Gene Targets to Consider
Not only specific metabolic steps, but compartmentation needs to be considered
Sarah Smith
Controlled Expression Overexpression Knockdowns Fluorescent Tagging
Diatoms as a System to Investigate Genetic Manipulation
Approaches To Improve Productivity
1. A versatile set of genetic manipulation tools has been developed, bringing the manipulation technology
for diatoms on a par with other model organisms.
2. Gene silencing does not occur.
3. G+C content is near 50% and codon optimization is not necessary (but it helps).
4. Tools developed in one species thus far have worked in other species.
Roshan Shrestha, Aubrey Davis, Luciano Frigeri
Characterization of CGI-58 Lipase Homologs:
Improved Lipid Content Plus a Non GMO Selection Approach Emily Trentacoste, Jennifer Hull, Bill Gerwick
Knockdowns exhibited a phenotype of increased TAG plus increased cell viability under Si-
limitation conditions.
Increased viability plus increased fluorescence (BODIPY and Chlorophyll) are selectable
phenotypes. Emily Trentacoste
Native Sequence, Non-GMO Selection Approach
Organisms are classified as GMOs only if DNA from another genus is introduced. Theoretically,
through recombination and cross-over such an arrangement could be generated by the native
host.
Transformants generated this way will not be classified as GMOs, can be selected, and may have
improved viability under stress than wild-type.
Additional genes can be manipulated by co-transformation.
Key Finding: Application of genetic manipulation approaches has led to the possibility
of native sequence-based metabolic engineering for field applications with no regulatory
issues.
promoter nat1 antisense fragment
terminator promoter antisense fragment
terminator
promoter antisense fragment
terminator
Transform and Select
Summary
Transcript-level changes are generally accurate indicators of metabolic changes.
Gene targets for metabolic engineering can be identified by transcriptomics analysis.
Manipulations affecting a variety of metabolic processes improved TAG accumulation
with no detriment to growth.
Improvements were substantial.
Genetic manipulation approaches led to the possibility of manipulations with non-
GMO classification.
Raffaela Abbriano
Orna Cook
Aubrey Davis
Mark Hildebrand
Jennifer Hull
Kalpana Manandhar-
Shrestha
Eva Sanchez
Roshan Shrestha
Sarah Smith
Benoit Tesson
Jesse Traller
Emily Trentacoste
Andrew Allen
Steve Federowicz
Trina M. Norden-Krichmar
Matteo Pellegrini
Sabeeha Merchant
Richard Sayre